The invention will be generally discussed in relation to substrates for use in manufacture of DNA arrays, of the type generally known as DNA chips, on substrates particularly planar substrates but the invention is not limited to that particular application but has wider ramifications and the invention is not intended to be limited to the manufacture of such DNA chips. Other physical or chemical reactions include manufacture of printed circuits, flat panel displays, semiconductor chips, nanotechnology, micro-electromechanical systems, flexible printed circuits, protein chips and lab-on-a-chip microfluidics.
In its broadest form the invention relates to substrates which can be used for spatially defined deposition of any of a wide variety of chemical substances onto the substrates. Substances may include, but are not limited to, coloured materials, dyes, metals, drug molecules, polymers, catalysts, anti-wetting agents, pigments, etching chemicals, layerings and reagents for de-blocking, blocking, derivatisation and activation of solid phase chemical groups. Arrays can include deoxyribonucleic acids (DNA), peptides, peptidenucleic acids (PNA), ribonucleic acids (RNA) and other solid phase chemical arrays and arrays assembled by combinatorial chemistry.
In general the manufacture of DNA chips involves the selective and sequential addition onto a substrate, of molecular units each with a protective group which is removed when the next molecular unit is to be added. One such method of manufacturing DNA arrays uses a process known as the phosphoramidite process which uses a trityl group or derivatives of the trityl group as the protective group. The invention is not limited to this process but will be discussed with respect to it.
The phosphoramidite process is a repetitive four stage process (deprotection, coupling, capping and oxidation) for the chemical synthesis of polymers particularly sequences of DNA oligonucleotides to form portions of DNA.
In the phosphoramidite process, a portion of DNA in single stranded form is built up by the sequential addition of one of the four nucleotides (coupled in phosphoramidite form) being the four components which make up DNA, the A, T, G and C nucleotides. Each terminal nucleotide has a chemically removable protecting group on it. A chemical reagent known as a de-protecting agent removes the protecting group exposing a reactive hydroxyl group and in the next stage a nucleoside (in phosphoramidite form) is coupled to the growing DNA string. The next stage is a capping step where any DNA strings which were de-protected but to which a nucleotide was not coupled are permanently capped to prevent unwanted nucleotides from adding to that molecule in later coupling steps. In the final step, oxidation of the newly formed inter-nucleotide phosphite linkage is carried out to convert the linkage to a phosphotriester. Typically, the de-protecting agent is dichloroacetic or trichloroacetic acid.
In the manufacture of DNA arrays, a number of different sequence DNA strands are built up on a substrate to enable biochemical analysis to take place. In this process it is necessary to selectively de-protect various portions, termed features, of the array and it is particularly to the requirement for this selective de-protecting that one particular embodiment of substrate of the present invention is directed.
Selective de-protecting by direct light activated chemistry or photo-removable de-protecting techniques has been developed but these are somewhat inefficient resulting in short solid phase oligodeoxynucleotides, 20 to 25 nucleotides in length, in rather large unit feature sizes of 10 to 50 microns and it is an object of this invention to provide a more efficient chemical de-protecting process.
The applicant has surprisingly found that by the use of electrically charged emulsions which include the chemical de-protecting agent in the discontinuous phase and which are selectively deposited on predefined areas of a planar or other shaped substrate under the influence of an electric field, then more accurate, localised and efficient de-protecting may be possible.